Photoacoustic Selective Plane Illumination Microscopy

The photoacoustic effect (PA) is a physical phenomenon involving the generation of sound waves following light absorption in a material sample. The photon absorption and subsequent non-radiative relaxation of the chromophores induces a rapid isochoric heating. Thus, it increases the pressure within the sample inducing a thermoelastic expansion, and the emission of a pressure wave called a photoacoustic wave 1. By utilizing low ultrasonic scattering, PA imaging enables high-resolution, deeply penetrating imaging in biological tissue. The base of the contrast in photoacoustic imaging is the different absorption coefficients of tissue components or suitable transgene labels in the sample. However, since transgene probes are few and they have a poor efficiency, there are few applications to living animals and processes at the cellular and subcellular levels. Reversibly switchable fluorescent proteins (rsFPs) have had a revolutionizing effect on life science imaging due to their contribution to optical nanoscopy as agents able to improve contrast-to-noise ratio and spatial resolution 2. Since PA requires pulsed illumination and depends on signal generation via nonradiative energy decay channels, rsFPs optimized for fluorescence imaging may not be ideal for PA due to competitiveness between light emission and heating 3. The aim of this project is the development of a novel approach in photoacoustic microscopy working both on the engineering of probes and setup. The probes belong to two different families of photochromic proteins: GAF3 4, and two novel mutants of GFPs obtained adding a fluorescence-decreasing mutation to wildQ/wildQT proteins 5. We will apply novels rsFPs in a brand new photoacoustic system based on Selective Plane Illumination Microscope (paSPIM).